Myelodysplastic syndromes (MDS) are a heterogeneous group of blood disorders characterized by ineffective and dysplastic hematopoiesis. There are few effective treatments for MDS, due in part to our incomplete understanding of the molecular basis of this disease. The recent discovery of high-frequency mutations affecting the RNA splicing machinery in MDS presents a significant opportunity to further our knowledge of MDS biology and inform the development of new therapeutics. However, the molecular consequences of spliceosomal mutations ARE unknown, hindering efforts to understand how these mutations contribute to dysplastic hematopoiesis and lead to new therapeutic opportunities. To address this gap in knowledge, we propose to determine the mechanistic, functional, and therapeutic consequences of mutations affecting the spliceosomal gene U2AF1, one of the most commonly mutated genes in MDS. We have built a team with experience in RNA splicing mechanisms and splicing-based therapeutics (Bradley), as well as MDS biology and patient care (Ramakrishnan, Shimamura). In Aim 1, we will determine how MDS-associated mutations alter U2AF1's normal role in 3' splice site recognition, including causing sequence-specific alterations in U2AF1:RNA interactions. In Aim 2, we will identify mRNAs that are mis-spliced in cells with U2AF1 mutations and subsequently translated into protein, and test the hypothesis that U2AF1 mutations give rise to molecular hallmarks of MDS cells. In Aim 3, we will test the hypothesis that chemical inhibition of splice site recognition wil selectively kill cells with U2AF1 mutations. At the conclusion of this study, we will have determined the mechanistic consequences of U2AF1 mutations for the splicing process, shown how these mutations contribute to molecular pathologies characteristic of MDS cells, and tested the potential of targeting the RNA splicing process itself as a new therapeutic avenue for MDS. Ultimately, we expect the proposed work to accelerate the pace at which new therapeutics may be developed to treat MDS.